**3.1 Organics conversion in anaerobic systems**

92 Biogas

materials with extremely different properties. These plastics are biodegradeable and are

This chapter intends to bring together the knowledge obtained from different applications of anaerobic technology in the treatment of various kinds of agro-industrial wastewaters to generate biogas. The first part covers essential information on the fundamentals of anaerobic technology, to demonstrate how the anaerobic treatment is able to generate significant volumes of methane-rich biogas. The wastewaters used in this chapter to generate biogas, contribute significantly in the pollution of the water bodies. In this opportunity the wastewater from Tequila vinasses were treated by different microbial consortia with energy purpose. This chapter illustrates the basics concepts of microbiology and biochemistry involved in the wastewater anaerobic treatment. The remainder focuses on various anaerobic reactor configurations and operating conditions used for the treatment of agroindustrial wastewaters different, show some examples with technical viability and the potential benefits that would be obtained by the utilization of the biogas as source of energy

Very old sources indicate that using wastewater and so-called renewable resources for the energy supply it is not new, it was already known before the birth of Christ. Even around 3000 BC the Sumerians practiced the anaerobic cleansing of waste. The Roman scholar Pliny described around 50 BC some glimmering lights appearing underneath the surface of

In 1776 Alessandro Volta personally collected biogas from the Lake Como to examine it. His findings showed that the formation of gas depends on a fermentation process and that may form an explosive mixture with air. The English physicist Faraday also performed some experiments with marsh gas and identified hydrocarbons as part of this. Around the year 1800, Dalton, Henry and Davy first described the chemical structure of methane, however the final chemical structure of methane (CH4), was first elucidated by Avogadro in 1821

In the second half of 19th century, more systematic and scientific in-depth research was started in France to better understand the process of anaerobic fermentation. The objective was simply suppress the bad odor released by wastewater pools. During their investigations, researchers detected some of the microorganisms which today are known to be essential for the fermentation process. It was Béchamp who identified in 1868 that a mixed population of microorganism is required to convert ethanol into methane, since several end products were formed during the fermentation process, depending on the

In 1876, Herter reported that acetate found in wastewater, stoichiometrically form methane and carbon dioxide in equal amounts. Louis Pasteur tried in 1884 to produce biogas from horse dung collected from Paris roads. Together with his student Gavon he managed to produce 100 L methane from 1 m3 dung fermented at 35ºC. Pasteur claimed that this production rate should be sufficient to cover the energy requirements for the street lighting of Paris. The application of energy from renewable resources started from this time on

used in the production of bioplastics (Mu et al., 2006) and other biochemicals.

to full scale.

**2. Historical background** 

swamps (Lee et al., 2010).

(Horiuchi et al., 2002).

characteristic of substrate (Lee et al., 2010).

(Deublein and Steinhauser, 2008).

The anaerobic digestive process is a natural biological process in which an interlaced community of bacteria cooperates to obtain a stable and auto-regulated fermentation through assimilation, transformation and decomposition of the residual organic matter present in waste and wastewater into biogas. This is a complex multistep process in terms of chemistry and microbiology, where the organic material is degraded to basic constituents to obtain methane gas under the absence of an electron acceptor such as oxygen. The common metabolic pathway and process microbiology of anaerobic digestion is shown in Fig. 1 (Khanal, 2008).

Generally, the anaerobic digestion process consists of four stages; the first one is called hydrolysis (or liquefaction), it consists in the transformation of complex organic matter such as proteins, carbohydrates and lipids into simple soluble products like sugars, long-chain fatty acids, amino acids and glycerin, this stage is carried out by the action of extracellular enzymes excreted by the fermentative (group 1) (Khanal, 2008).

In the second step, called the acidogenic stage fermentative bacteria use the hydrolysis products to form intermediate compounds like organic acids, including volatile fatty acids (VFA). Theses VFA along with ethanol are converted to acetic acid, hydrogen and carbon dioxide by other group of bacteria known as hydrogen-producing acetogenic bacteria (group 2) (Khanal, 2008).

Organic acids are oxidized partially by bacteria called acetogenic in the third stage, which produce additional quantities of hydrogen and acetic acid. The acetogenesis is regarded as thermodynamically unfavorable unless the hydrogen partial pressure is kept below 10-3 atm, pathway efficient removal of hydrogen by the hydrogen-consuming organisms such as hydrogenotrophic methanogens and/or homoacetogens (Zinder, 1988).

Finally, in the fourth stage, both acetic acid and hydrogen are the raw material for the growth of methanogenic bacteria, converting acetic acid and hydrogen to biogas composed mainly of methane, carbon dioxide and hydrogen sulfide (Khanal, 2008).

Acetate, H2 and CO2 are the primary substrate for methanogenesis. On chemical oxygen demand (COD) basis about 72% of methane production comes from the decarboxylation of acetate, while the remainder is from CO2 reduction (McCarty, 1964). The groups of microorganisms involved in the generation of methane from acetate are known as acetotrophic or aceticlastic methanogens (group 3). The remaining methane is generated

Biogas Production from Anaerobic Treatment of Agro-Industrial Wastewater 95

a. **Fermentative Bacteria (group 1):** This group of bacteria is responsible for the first stage of anaerobic processes. The anaerobic species belonging to the family of Streptococcaceae and Enterobacteriaceae and the genera of *Bacteroides, Clostridium, Butyrivibrio, Eubacterium, Bifidobacterium* and *Lactobacillus* are most commonly involved

b. **Hydrogen-Producing Acetogenic Bacteria (group 2):** This group of bacteria metabolizes higher organic acids (propionate, butyrate, H2, etc.), ethanol and certain aromatic compounds (i.e. benzoate) into acetate, H2 and CO2 (Zinder, 1998). The anaerobic oxidation of these compounds is not favorable thermodynamically by hydrogen-producing bacteria in a pure culture, however in a coculture of hydrogenproducing acetogenic bacteria and hydrogen-consuming methanogenic bacteria, these exists a symbiotic relationship between these two groups of bacteria. It is important to point out that during anaerobic treatment of complex wastewater such as vinasses or slaughterhouse, as many as 30% of the electrons is associated with propionate oxidation. Thus, these chemical appears to be more critical than oxidation of other

c. **Homoacetogens Bacteria (group 3):** Homoacetogenesis has attracted much attention in recent years because of its final product acetate, an important precursor to methane generation. The responsible bacteria are either autotrophs or heterotrophs. The autotrophic homoacetogens utilize a mixture of hydrogen and carbon dioxide, with CO2 serving as the carbon source for cell synthesis. The heterotrophics homoacetogens, on the other hand, use organic substrate such as formate and methanol as a carbon source

CO2 + H2 CH3COOH + 2H2O (1)

4HCOOH CH3COOH + 2CO2 + 2H2O (3)

 4CH3OH + 2CO2 3CH3COOH + 2CO2 (4) *Acetobacterium woodii* and *Clostridium aceticum* are the two mesophilic homoacetogenic bacteria isolated from sewage sludge (Novaes1986). Homoacetogenic bacteria have a high thermodynamic efficiency; as result there is no accumulation of H2 and CO2 during growth

d. **Metanogenic Bacteria (group 4 and 5):** Methanogens are obligate anaerobes and considered as a rate-limiting specie in anaerobic treatment of wastewater. Abundant methanogens are found in anaerobic environments rich in organic matter such as swamps, marches, ponds, lake and marine sediments, and rumen of cattle. Most methanogens can grow by H2 as a source of electrons via hydrogenase as shown in the

 4H2 + CO2 CH4 + 2H2O (5) The source of H2 is the catabolic product of other bacteria in the system, such as hydrogenproducing fermentative bacteria, especially *Clostridia* (group 1) and hydrogen-producing acetogenic bacteria (group 2). The hydrogenotrophic pathway contributes up to 28% of the

4CO + 2H2O CH3COOH + 2CO2 (2)

organic acids and solvents (Deublein and Steinhaunser 2008).

while producing acetate as the end product (Eq. 1 to 4) (Khanal, 2008).

in this process (Novaes, 1986).

on multicarbon compounds (Zeikus 1981).

follow reaction (Eq. 5) (Khanal, 2008):

from H2 and CO2 by the hydrogenotrophic methanogens (group 4). Since methane is largely generated from acetate, acetotrophic methanogenesis is the rate-limiting step in anaerobic wastewater treatment. The synthesis of acetate from H2 and CO2 by homoacetogens (group 5) has not been widely studied. Mackie and Bryant (1981) reported that acetate synthesis through this pathway accounts for only 1-2% of total acetate formation at 40°C and 3-4% total solids at 60°C in a cattle waste digester.

Fig. 1. Steps of anaerobic digestion of complex organic matter (the number indicate the group of bacteria involved in the process).
